1. Field of the Invention
The present invention relates to a method of coupling binding agents to a substrate surface by passing a binding agent-containing fluid flow over the surface, and more particularly by using hydrodynamic addressing techniques to selectively direct fluid flows to desired surface areas. The invention also relates to analytical use of the method.
2. Description of the Related Art
Flow cells are used extensively nowadays in a variety of analytical systems. Typically, the flow cell has an inlet opening, a flow channel with a sensing surface, and an outlet opening. A sample fluid to be investigated is introduced through the inlet opening, passes through the flow channel and leaves the flow cell through the outlet opening. The flow cell may have more than one inlet opening and optionally more than one outlet opening to permit desired manipulations of the flow pattern within the flow cell.
The sensing surface usually comprises a substance layer to which a recognition element for an analyte in the sample is immobilised, typically a biochemical affinity partner to the analyte. When the analyte interacts with the recognition element, a physical or chemical change is produced on the sensing surface that can be detected by a detector, e.g., an optical, electrochemical or calorimetric detector. A flow channel may contain two or more sensing surfaces with different recognition elements.
The sensing surface or surfaces in the flow cell may be functionalized, or sensitized, in situ, i.e., within the flow cell. WO 90/05305 discloses a method for functionalising a sensing surface having functional groups thereon by passing a reagent solution containing a bi- or polyfunctional ligand over the surface, the ligand having a function which immobilizes the ligand on the sensing surface and at least one more function which is exposed on the sensing surface for interaction with the analyte. In a specific embodiment, the sensing surface has a bound carboxymethyl-dextran layer. After activation of the surface through derivatization with N-hydroxysuccinimide, mediated by N-ethyl-N′-(dimethylaminopropyl)carbodiimide, a ligand in the form of aminotheophylline or aminobiotin is coupled to the activated surface.
WO 99/36766 discloses methods and systems using hydrodynamic addressing techniques to allow immobilization of different ligands to discrete sensing areas within a single flow cell channel, as well as to permit controlled sample delivery to such sensitized areas. For a Y-type flow cell, which has an inlet end with two inlet ports and an outlet end with one outlet port, and a sensing surface between the ends, WO 99/36766 describes sensitization of two spaced apart sensing areas with different ligands. This is done by providing a laminar flow of a sensitising fluid adjacent to a laminar flow of non-sensitizing (blocking) fluid such that the fluids flow together over the sensing surface with an interface to each other. By adjustment of the relative flow rates of the two fluids the interface may be positioned laterally such that sensitising fluid selectively contacts a desired area of the sensing surface. More specifically, if the sensitizing fluid initially contains a first ligand capable of binding to the sensing surface and the interface is positioned such that the sensitizing fluid covers, say, about one third of the lateral extension of the sensing surface, and the blocking fluid covers the remaining two thirds, the first ligand will be immobilized to the first-mentioned third of the sensing surface. Then, the sensitizing fluid is replaced by blocking fluid, and the blocking fluid is replaced by a sensitizing fluid containing a second ligand. By positioning the interface such that the sensitizing fluid again covers about one third of the lateral extension of the sensing surface, now, however, at the opposite side of the flow path, the second ligand will be immobilized to that area, thereby providing a sensing surface which, as seen laterally, has about one third immobilized with the first ligand, about one third immobilized with the second ligand, and an intermediate non-immobilized third which only has been in contact with blocking fluid and may suitably be used as a reference area.
For providing more than two differently sensitized sensing surface areas, WO 99/36766 discloses the use of a so-called ψ-type flow cell having three inlets and a single outlet. In this embodiment, a laminar flow of sensitizing fluid is sandwiched between two laminar flows of blocking fluid, and the sensitizing fluid may thereby be displaced laterally to selectively contact a number of sensing surface areas.
A similar use of a ψ-type flow cell is disclosed in WO 00/56444.
While the ψ-type flow cell is advantageous in comparison with the Y-type flow cell in that the former readily permits sensitization with more than two different ligands, the ψ-type flow cell requires the use of an additional pump (one pump for each fluid flow inlet), which considerably complicates the control of the different laminar flows. It would therefore be desired to be able to use a Y-type flow cell to sensitize a sensing surface with more than two different ligands.
Accordingly, it is an object of the present invention to provide a method which permits coupling of multiple different ligands, or generally binding agents, to respective surface areas by hydrodynamic addressing using only two adjacent laminar fluid flows, such as e.g., in a Y-type flow cell.